The development of type 2 diabetes and its associated cardiovascular complications has been linked to a failure to adequately expand adipose tissue mass. Recent studies have demonstrated that patients with insulin resistance have a defect in adipogenesis and the mechanisms leading to this are still poorly understood. In this proposal we will investigate the potential role of lipid signals in the generation of this adipogenic defect. Our previous work was targeted towards the understanding of an extreme phenotype of insulin resistance characterized by the absence of fat: congenital generalized lipodystrophy secondary to the AGPAT2 mutation. We determined that the defect in adipogenesis was secondary to a faulty activation of the PI3K/Akt pathway and the master regulator of adipogenesis, PPAR. AGPAT2 is part of the triglyceride (TAG) synthesis pathway, and another enzyme in this pathway, GPAT3, also regulates adipogenesis. Thus the utilization of the TAG synthesis pathway would be an efficient way for preadipocytes to detect increased flux of fatty acids and produce intermediates that would stimulate adipogenesis. Indeed lipid intermediates with the capacity to regulate adipogenesis can be generated by this pathway (i.e. cyclic phosphatidic acid as a PPAR antagonist). We hypothesize that individuals with an enhanced intrinsic capacity for generating pro-adipogenic signals (lysophosphatidic acid vs. cyclic phosphatidic acid) in the face of increased fatty acid supply are protected from obesity-induced insulin resistance. To test this hypothesis we plan to first determine effects of different enzymes from the TAG synthesis pathway in the generation of bioactive lipids capable of regulating adipogenesis. In the second specific aim we will determine effects of TAG synthesis enzymes on the adipogenic impairment associated with exposure to a high concentration of nutrients. In the third specific aim we will determine if preadipocytes from obese insulin-resistan individuals have a defect in the generation of pro-adipogenic lipid intermediates derived from the TAG synthesis pathway when compared to obese insulin sensitive individuals. To accomplish this we will compare steady state levels of lipid intermediates from the TAG pathway and the rate of labeling through the TAG synthesis pathway utilizing 13C labeled fatty acids. At the completion of these studies we will have defined the faulty lipid signal present in obese insulin-resistant individuals. PUBLIC HEALTH RELEVANCE: Patients predisposed to the development of type 2 diabetes and heart disease have a more limited capacity of storing fatty acids which are then 'spill over' other tissues. This causes the toxic effects typically seen in these conditions. This proposal will examine the cause underlying the defect in storage capacity of adipose tissue.